Cell surface receptors play an important role in biology by relaying extracellular messages into intracellular signaling pathways, thereby allowing individual cells to appropriately respond to their surroundings. Such receptors play critical roles in processes including growth control, developmental patterning, hormonal signaling and the immune response. The function of such receptors is thus frequently involved in diseases and conditions involving these processes such as cancer, developmental defects, endocrine disorders, tissue rejection, and autoimmune dysfunctions. These same receptors are also frequently involved in the infection of a host by a pathogenic organism. In particular, many viruses utilize such receptors to facilitate entry into a host cell. These diseases, conditions, and infections are thus amenable to treatment by compounds which are capable of blocking a receptor""s function as a mediator of signal transduction or pathogenic intrusion.
Ultimately, there are widespread medical applications for such receptor blocking functions owing to this correspondingly widespread involvement of receptors in organismal function. For example, the recruitment of leukocytes from the circulation to sites of injury and infection is a key process in the physiological response to wound healing and the clearance of pathogenic organisms (Springer (1994) Cell 76: 301-14). Recent advances in the understanding of the molecular mechanisms that regulate leukocyte recruitment have identified a complex interplay between leukocyte, cytokines, chemokines, adhesion molecules, and extracellular matrix components that is essential for directed leukocyte migration. Chemokines comprise an ever enlarging family of small molecular weight cytokines that play a key role as effector molecules which stimulate leukocytes to leave the circulation and migrate to the sites of inflammation and injury. This superfamily of cytokines has well over thirty distinct member, which bind to subsets of G-protein coupled serpentine receptors. Despite the beneficial properties that chemokines have in the wound healing process and for the clearance of infectious organisms, they also can have pathophysiological consequences. Continued expression of chemokines stimulates the accumulation of leukocytes which, when appropriately activated, release injurious enzymes and oxidative radicals. Many inflammatory and immunological disorders, such as arthritis, asthma, reperfusion injury, and atherosclerosis, are characterized by increased levels of specific sets of chemokines. Therefore, a likely target for suppression of inflammatory or immunological disorders is to inhibit chemokine expression or function, thereby limiting the degree of leukocyte infiltration.
Complement activation also plays a fundamental role in inflammation. Inappropriate or excessive activation of the complement system can lead to harmful, potentially life-threatening consequences due to severe inflammatory tissue destruction. These consequences are clinically manifested in various disorders, including septic shock, multiple organ failure and hyperacute graft rejection. In addition, inappropriate activation of the inflammatory response is associated with immune complex and/or autoimmune diseases such as glomerulonephritis and systemic lupus erythematosus (SLE). Such diseases may be triggered by deficiencies in the ability to solubilize and clear circulating immune complexes, leading to the accumulation and deposition of such complexes in blood vessel walls and tissues where they activate the complement cascade and produce local inflammation. Furthermore, such undesirable inflammatory reactions may subsequently augment the antigen presenting functions of mononuclear phagocytes, leading to the abnormal presentation of self antigensxe2x80x94a condition known as autoimmunity. Thus strategies to antagonize components of the complement system would potentially have far ranging applications in the treatment of inflammatory and autoimmune dysfunctions. Indeed, genetic complement deficiencies or complement depletion have been proven to be beneficial in reducing tissue injury in a number of animal models of severe complement- dependent inflammation (Kirschfink M (1997) Immunopharmacology (Netherlands) 38: 51-62).
The complement system relies upon the function of a number of cell surface receptors. These include complement receptor 1 (CR1, also known as C3b Receptor and CD35), complement receptor type 2 (CR2, also known as C3d Receptor and CD21), and complement receptor type 3 (CR3, also known as CR3. MAC-1 and CD11bCD18). Each of these receptors serves a unique function in complement mediated immune response, and so agents designed to antagonize each of these receptors have unique therapeutic benefits. Indeed, a genetically engineered, soluble form of CR1, lacking transmembrane and cytoplasmic domains, has been tested as an anti-inflammatory agent and found to limit tissue injury in an in vivo model of acute inflammation. Another strategy for treating inflammatory disorders is to interfere with complement receptor 3 (CR3, CD18/11b)xe2x80x94mediated adhesion of inflammatory cells to the vascular endothelium. Experimental therapies which target complement receptor function also include the administration of CR3-specific antibodies which interfere with receptor-mediated adhesion of inflammatory cells to the vascular endothelium (Kirschfink, M. et al. (1997) Immunopharmacology (Netherlands) 38: 51-62). Such studies have demonstrated that protection against complement-mediated inflammatory tissue damage can be achieved using complement receptor antagonists in various animal models of sepsis, myocardial as well as intestinal ischemia/reperfusion injury, adult respiratory distress syndrome, nephritis and graft rejection. Thus complement receptor antagonists are suitable therapeutic agents to control inflammatory diseases and inflammatory related conditions.
Cell surface receptors have also been implicated in the genesis of cancer through the molecular analysis of the etiology of this group of related diseases characterized by a loss of cellular growth control. Indeed, cell surface receptors play multiple roles in oncogenic transformation including: serving as portholes for cellular infection by known and suspected human tumor viruses including Epstein-Barr virus, human T-cell leukemia virus (HTLV), Hepatitis B virus, and Papilloma viruses; serving as mediators of growth factor and transforming growth factor dependent stimulation of cancer cell growth through autocrine or paracrine mechanisms; and, finally, by indirectly facilitating the progression of cancer by potentiating the vascularization of tumor tissue and thus allowing for the continued growth of a mass of oncogenically transformed cells. Therapeutic agents capable of blocking any or all of these receptor functions thus have great utility in the treatment and prevention of human cancers. For example, the mitogenic action of epidermal growth factor (EGF) is mediated by ligand-induced autophosphorylation of the EGF receptor (EGFR), and EGFR is commonly overexpressed in solid human tumors. Compounds designed to block receptor tyrosine kinase activity by serving as competitive inhibitors of ATP binding to the intracellular kinase catalytic domain of the EGFR, have been shown in cell culture studies to be capable of blocking EGF-stimulated growth in a concentration dependent manner without affecting basal growth (Wakeling et al. (1996) Breast Cancer Res. Treat. (Netherlands) 38: 67-73; see also Rewcastle et al. (1998) J. Med. Chem. 41:742-51). Furthermore, a broad spectrum neuropeptide receptor antagonist has been shown to inhibit tyrosine kinase activation, block cell growth and increase apoptosis in in vitro and in vivo studies of a small cell lung cancer in which cell growth is sustained by multiple autocrine and paracrine growth loops involving bombesin-like neuropeptide growth factors (Tallett, et al. (1996) Cancer Res. 56: 4255-63). Indeed, for certain cancers generally unaffected by conventional chemotherapeutics, such as colorectal cacrcinomas, the targeting of growth factor receptors appears to be a logical approach to designing effective treatments (reviewed in Normanno et al. (1998) Cancer Detect. Prev. 22: 62-7).
In another example of the involvement of cell surface receptors in human cancer, paracrine signaling by interleukin 1 (IL-1) and tumor necrosis factor (TNF) is known to trigger the proliferation of human cervical carcinoma-derived epithelial cells and this process is mediated by an induced autocrine interaction of secreted amphiregulin with EGFR. In this system a number of different receptor antagonists, including an IL-1 receptor antagonist, soluble TNF type 1 or 2 receptor extracellular. domains, or EGFR monoclonal antibodies, prevented mitogenic stimulation (Woodworth et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92: 2840-4). These results demonstrate that cell surface receptors responding to both paracrine and autocrine signals are involved in oncogenic transformation and, furthermore, that receptor antagonists targeting either type of growth factor signaling can serve as effective oncogenic growth inhibitors. Similarly, dopamine receptor antagonists inhibited the growth of human small cell lung cancer in tumor implanted athymic nude mice (Ishibashi et al. (1994) Cancer Res. 54: 3442-6); and steroid receptor antagonists have proven valuable in the treatment of certain androgen-dependent cancers (reviewed in Wakeling (1992) Cancer Surv. (United States) 14: 71-85).
Furthermore, later stages in tumor establishment and metastasis are also potential targets for receptor antagonist therapeutics. For example, recent studies on angiogenesis in human solid tumors have suggested that inhibiting the function of vascular endothelial growth factor (VEGF) receptors and basic fibroblast growth factor (bFGF) receptor would prove effective in blocking neovascularization in a variety of tumor types (reviewed in Toi et al. (1998) Gan To Kagaku Ryoho (Japan) 24: 2202-6). In addition, the urokinase plasminogen activator (uPA) and its receptor are essential mediators of such metastatic functions as extracellular matrix proteolysis and tumor cell migration and small molecule antagonists of the uPA receptor promise to serve as useful adjuvants in combination with existing chemotherapy strategies (Ignar, et al. (1998) Clin. Exp. Metastasis (England) 16: 9-20). Thus it is clear that receptor antagonists can inhibit the initiation and progression of cancer by blocking any of a number of processes involved in oncogenic transformation and growth. Indeed the use of receptor antagonists has even proven fruitful in the prevention of nausea and emesis (vomiting) in cancer patients undergoing chemotherapy. In particular, antagonists of the 5-hydroxytryptamine 3-receptor have proven effective toward this goal (Perez et al. (1998) Cancer J. Sci. Am. 4: 52-8).
The utility of cell receptor antagonists is not limited to the treatment of cancers and inflammatory diseases. For example, many examples of pharmacologically active receptor antagonist compounds exist in the art. Cell surface receptors on neurons mediate neurotransmitter signaling and so neurotransmitter receptor antagonists have applications in treating neurological diseases and conditions as well mental and emotional disorders. For example, recent evidence suggests that Parkinson""s disease is principally a glutamate hyperactivity disorder and hence treatable with glutamate receptor antagonists (reviewed in Starr (1995) Synapse 19: 264-93). And the usefulness of neurotransmitter receptor antagonists in treating mental and emotional conditions is also well establishedxe2x80x94for example the use of tricyclic muscarinic receptor antagonist compounds like lofepramine to treat depression (reviewed in Leonard (1987) Int. Clin. Psychopharmacol. (England) 2: 281-97). Furthermore, receptor antagonists have been used successfully to treat neuroendocrine disorders such as in the case of hyperthyroidism treatments employing beta-blocker compounds which antagonize the beta-receptor mediated effects of catecholamines (reviewed in Geffner and Mershman (1992) Am. J. Med. 93: 61-8).
Still other examples of receptor antagonists which are useful as therapeutic agents include angiotensin II receptor antagonists. Interruption of the renin-angiotensin-aldosterone system by such compounds has been shown to dramatically reduce renal damage in a renal hypertensive disease model (reviewed in Ibrahim, et al. (1998) Semin. Nephrol. 17: 431-40). Examples of widely used therapeutic receptor antagonists include histamine 2 receptor-antagonists which are sold over-the-counter for use in treating acid peptic disorders including gastric ulcers, duodenal ulcers, and gastroesophageal influx disease (Sanders (1996) Clin. Ther. 18: 2-34).
Receptor antagonists are also capable of altering the ability of infectious agents such as viruses and microbes (including pathogenic bacteria and fungi) to recognize and utilize host cell surface receptors. Indeed, as mentioned earlier, there are several examples of tissue-tropic tumor causing viruses, including human T-cell leukemia virus (HTLV), Epstein-Barr virus, hepatitis B virus and various human papillomaviruses, which are known or suspected of using specific host cell surface receptors as portholes of entry into a host cell. Significantly, another well examined virus, HIV (Human Immunodeficiency Virus), also frequently results in cancer in the afflicted individual. In the case of HIV, however, cancer is thought to result indirectly from, for example, the virus""s weakening of the immune system, leaving the patient susceptible to viruses such as the above-mentioned tumor-causing Epstein-Barr virus. Indeed, infection by HIV also frequently leads to acquired immunodeficiency syndrome (AIDS), which commonly includes the development of Kaposi""s sarcomaxe2x80x94a type of tumor which may be induced by growth factors released by HIV-infected cells. Thus appropriate receptor antagonists which target either the cell surface receptors implicated in HIV host cell infection or other cell surface receptors involved in the progression of AIDS (e.g. these receptors responding to Kaposi""s sarcoma-associated secreted growth factors), would provide valuable therapeutic benefits in preventing HIV infection and treating AIDS.
Indeed, due to the public health concern and ensuing intense research, much is known about the mechanism by which HIV exploits host receptors to gain entry into a target cell. HIV-1 and related viruses possess a virion envelope glycoprotein (gp 120/41) which interacts with at least two cellular receptors: The CD4 molecule and a seven-transmembrane domain G-protein coupled chemokine receptor (D""Souza M. P. and Harden V. A. (1996) Nature Med. 2: 1293-1300). Macrophage-tropic (M-tropic) strains of HIV-1 replicate in macrophages and CD4+ T cells and use the CC chemokine receptor CCR5 (Dragic, T. et al. (1996) Nature 381: 667-73; Deng, H. et al. (1996) Nature 381: 661-6; Alkhatib, G. et al. (1996) Science 272: 1955-8; Doranz, G. J. et al. (1996) Cell 85: 1149-58; Choe, H. et al. (1996) Cell 85: 1135-48). These HIV-1 viruses can be classified as R5 type based on their co-receptor usage (Berger, H. A. et al. (1998) Nature 391:240). The CCR5 co-receptor is used by almost all primary HIV-1 isolates regardless of viral genetic subtype, and by the related lentiviruses HIV-2 (Berger, H. A. et al. (1998) Nature 391: 240) and simian immunodeficiency virus (SIV) (Edinger, A. L. et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4005-10). T-tropic isolates of HIV-1 replicate in primary CD4+ T cells, as well as established CD4+ T cell lines and macrophages. All of these viruses use the CXC chemokine receptor CXCR4, and many of them also use CCR5 (Feng, Y. et al. (1996) Science 272: 872-7; Simmons, G. et al. (1996) J. Virol. 70: 8355-60; and Connor, R. I. (1997) J. Exp. Med. 185: 621-8). Those viruses that only use CXCR4 are referred to as X4, whereas viruses that use both receptors with comparable efficiency are referred to as R5X4 (Berger, H. A. et al. (1998) Nature 391: 240). Although CCR5 and CXCR4 are believed to be the primary receptors for entry of HIV-1, nine additional chemokine receptors, including one encoded by cytomegalovirus have been shown by in vitro assays to serve as co-receptors for HIV and SIV. Within the human genome, there exist approximately fifty additional open reading frames with sequence similarity to chemokine receptors; some of these may ultimately contribute to the growing list of chemokine receptors with HIV co-receptor activity (Cairns, J. S. and D""Souza, M. (1998) Nature Med. 4:563-8).
Several strategies have been devised to interfere with the progression of HIV-1 infection by preventing the expression or function of chemokine receptors. Such treatments include those employing administration of CD4+T cells which have been treated with antibodies to induce down-regulation of CCR5 expression and those employing genetic manipulation of host cells so that they express protective genes which interfere with cytokine receptor protein expression or localization. Examples of protective genes of the latter example include those encoding genetically engineered ribozymes designed to recognize and cleave cytokine-encoding mRNAs, those encoding intrabodies or intracellular antibodies designed to bind to chemokine receptors and interfere with their expression, and those encoding intrakines which are secretion-defective forms of natural cytokines which remain intracellular and bind to newly synthesized cytokine receptor proteins and trap them in the endoplasmic reticulum where they are rapidly degraded. Other strategies have been devised to interfere with the extracellular recognition of the HIV-1 virion gp120, as opposed to the cell surface expression, of cytokine receptors. For example, monoclonal antibodies have been developed to target chemokine receptors. The first such reagent was 12G5, a murine mAb against the CXCR4 receptor (Endres, et al. (1997) Cell 87:745-56). Of the mAb to CCR5, one particular murine mAb designate 2D7, completely blocked the binding and chemotaxis mediated by the chemokines RANTES, MIP-1xcex1, and MIP-1xcex2, This mAb also efficiently blocked the infectivity of several R5 and R5X4 viruses. Mapping studies have revealed that 2D7 maps to the second extracellular loop of CCR5, an important domain for both gp120 and chemokine binding (Wu et al. (1997) J. Exp. Med. 186: 1373-81). However, the development of mAbs for use as anti-HIV therapeutics is still subject to several obstacles including the high cost of production of these reagents, accessibility of the targeted cell population, and the possible immunogenicity of the mAb.
Another strategy for blocking HIV-1 infection has been to utilize natural chemokine receptor ligands which have been shown to compete with HIV-1 envelope glycoprotein for binding to HIV-1 chemokine coreceptors in certain instances. For example, experimental cell culture studies have demonstrated that RANTES, MIP-1a, and MIP-1b can inhibit the replication of R5 viruses, the CCR5 chemokine-utilizing subclass of HIV-1 viruses (Cocchi et al. (1995) Science 270: 1811-5). Similarly, the chemokine SDF-1 has been shown to competitively block viral entry of X4 viruses (Bleul et al. (1996) Nature 382: 829-33; Oberlin et al. (1996) Nature 382: 833-5). Other recently described CC chemokines with anti-viral properties include 1-309 the ligand for CCR8 (Tiffany et al. (1997) J. Exp. Med. 186: 165-70) and macrophage-derived chemokine (MDC) which exhibits a broad range of suppressive activity against diverse primate lentiviruses (Pal et al. (1997) Science 278: 695-8).
Several studies are planned or at the proof-of-concept stage using biologically active or inactive variants of these molecules. One such agent that has already been tested in the clinic is a variant of the CCR5-binding chemokine MIP-1xcex1, called BB-10010. In a phase I study of BB10010, no consistent changes were noted in viral load, CD4 counts, or HIV isolate co-receptor usage. This is the predicted result considering the median plasma concentration of BB-10010 was only 3.5 ng/ml after six days of treatment, much lower than the 90-900 ng/ml range required to see antiviral effects of chemokines in vitro. In the absence of innovative dosing strategies to improve the efficacy of BB-10010, this ligand is unlikely to succeed in the clinic.
Therapeutic interventions based on administration of chemokines or over expression of these bioactive compounds are also compromised because of the key role these molecules play in inflammation. Furthermore, other in vitro observations also complicate the therapeutic use of chemokines. One overriding concern is the observation that in certain circumstances, the xcex2-chemokines administered alone can actually enhance the replication of X4 HIV-1 isolates (Trikola, et al. (1998) J. Virol. 72: 396-404). Similarly, SDF-1, has been found to stimulate certain R5 HIV-1 isolates (Trikola, et al. (1998) J. Virol. 72: 396-404). In both of these instances, the stimulatory effect seems to depend upon the ability of the chemokine to transmit intracellular signals to the target cell following interaction with its receptor on the target cell membrane. Thus compounds which possess the HIV-1 gp120 blocking activity of these chemokines, but which do not possess their ability to activate the chemokine receptor signal transduction cascade resulting in undesirable HIV-1 replication enhancement, would obviously be preferable as a therapeutic. A second concern is that under certain circumstances, MIP-1xcex1, MIP-1xcex2, and RANTES, when administered alone, can enhance (Schmidtmayerova et al. (1996) 382: 767) CCR5-mediated fusion, entry and replication of R5 strains in macrophages, in contrast to their inhibitory properties in T cells. Because macrophages are likely to be among the first cells exposed to HIV and constitute a reservoir for the virus, these observations engender caution, and suggest again a need for therapeutics which retain the ability to block HIV-1 gp120 interaction with the chemokine receptor, yet which do not possess the abovementioned undesirable activities.
Modified xcex2-chemokines that block HIV infection without inflammatory side effects or the HIV-1-stimulatory effects of the parent molecules are second generation compounds with such therapeutic promise. Two xcex2-chemokine derivatives that bind CCR5 and lack cellular signaling capabilities are under investigation: RANTES(9-68), a truncated form of RANTES (Arenzana-Seisdedos et al. (1996) Nature 383: 400); and aminooxypentane (AOP)-RANTES, a version that is chemically modified at the amino terminus (Simmons, et al. (1997) Science 276:276-9). Both of these compounds exhibit increased potency when compared to RANTES and inhibit the infection of primary lymphocytes by R5 viruses in tissue culture experiments without stimulating X4 subclass HIV-1 replication. AOP-RANTES was also able to inhibit replication of R5 strains in primary human macrophages. Although the unmodified xcex2-chemokines block HIV infection of dendritic cells (Granelli-Piperno et al. (1996) J. Exp. Med. 184: 2433-8), the inhibitory properties of these modified chemokines on dendritic cells and other sites of viral entry remain unknown. Two peptides that appear to block the CXCR4-HIV interaction are T22 (Murakami et al. (1997) J. Exp. Med. 186: 1389-93) and ALX40-4C (Doranz et al. (1997) J. Exp. Med. 186: 1395-1400). T22 is an 18-amino acid peptide derived from the hemocyte debris of the horseshoe crab. It blocks membrane fusion and infection by X4 type HIV-1 viruses as well as chemotaxis in response to SDF-1. ALX40-4C is a highly cationic peptide containing nine arginines. This compound also blocks HIV envelope interactions and SDF-1 interaction with CXCR4.
While these natural and synthetic chemokine-related compounds show some promise as anti-HIV therapeutics, their practical use presents several difficulties. In particular, such peptides in general are of uncertain stability in the bloodstream, and chemokines, in particular, are thought to possess a relatively short half-life in circulation ( less than 10 minutes) (Van Zee et al. (1992) J. Immunol. 148: 1746-52). At least one limitation to chemokine serum half-life appears to be their propensity for binding to cell surface proteoglycans as well as other glycosaminoglycans present in intercellular and extracellular matrices. This tendency reflects the apparent use by chemokines of heparan sulfate proteoglycans to promote efficient binding to chemokine receptors and to thereby promote the anti-viral activity of chemokines in cell culture experiments (Oravecz, et al. (1997) J. Immunol. 159: 4587-92). Furthermore, recent studies suggest that HIV-suppressive chemokines produces by CD8+ T cells (Cocchi, et al. (1995) Science 270: 1811-15) are secreted in a form complexed to proteoglycans (Wagner et al. (1998) Nature 391: 908-11).
As summarized above, the ability to block the function of a receptor with a receptor antagonist provides a means of specifically interfering with the molecular processes underlying many diseases, conditions, and infectious states. Nevertheless, several obstacles remain to the development of effective receptor antagonist therapeutics for treating inflammatory disorders, cancers, infections and other diseases and conditions whose etiology involves the function of a cell surface receptor. In particular many of the above- described receptor antagonists, while practical as experimental reagents for investigating the potential utility of targeting a particular receptor, are inappropriate as pharmaceutical therapeutics for various reasons such as poor solubility, toxicity, short serum half-life, or undesirable side effects. For example, although the initial laboratory data on the ability of the above-described natural and altered chemokine and chemokine derivative peptides to block the interaction of HIV envelope with chemokine receptors may be encouraging, the therapeutic potential of these compounds administered alone is uncertain owing to a number of likely problems confronting their use in vivo. In particular, the abovementioned problem of the short serum half-life of exogenously administered chemokines needs to be addressed. Another major problem with using chemokines as anti-HIV therapeutics is their previously mentioned tendency to increase viral replication under certain circumstances. Yet another problem with this potential HIV therapeutic is the occurrence of undesirable side-effects owing to the continued activation of chemokine responsive cells. Within the anticipated time frame required to effectively lower viral loads using such a treatment strategy, such chronic stimulation of the immune system would create additional medical complications for the HIV infected patient.
Furthermore, the development of effective receptor antagonist compounds for treating the abovementioned diseases and conditions is frequently limited by the complicated bioassays utilized to study such processes. Indeed such assays often preclude the use of high-throughput screening techniques to identify appropriate receptor antagonists compounds. Furthermore, antagonists identified through such techniques must undergo further in vivo screening to characterize adverse side effects and to minimize toxicity. Rational design strategies offer a solution to at least the first and frequently the second cited problem of developing receptor antagonist therapeutics. Unfortunately most rational design strategies require detailed knowledge of the structure of the therapeutic target. However the detailed three dimensional structure, as determined by X-ray crystallographic analysis, is frequently not available for such cell surface receptorsxe2x80x94at least in part owing to the difficulty in obtaining adequate crystals from lipophilic membrane proteins.
There is thus a great need for methods of formulating effective receptor antagonists based upon rational design concepts that do not require extensive screening.
The present invention affords a solution to this and other problems confronting the developer of suitable receptor-targeting therapeutics. In particular, in one aspect, the present invention provides for the facile modification of known receptor ligands with ligand binding molecules to form therapeutic receptor ligand-containing antagonist complexes. The invention thus allows for facile conversion of natural receptor agonists into novel pharmaceutical formulations which act as receptor antagonists.
Preferred receptor ligands are natural or synthetic molecules that bind to receptors involved with disease causing ligand/receptor-mediated signaling pathways or which are involved with extracellular recognition of an infectious agent. Examples of such receptors include: chemokine coreceptors, which mediate host cell uptake of viruses such as HIV, growth factors, which are associated with the development of certain cancers, and complement receptors, which are associated with the development of certain inflammatory diseases. Examples of preferred receptor ligands include: chemokines and receptor binding portions thereof; growth factors; and complement proteins. Particularly preferred chemokine ligands include interleukins, tumor necrosis factors, lymphokines, interferons and lymphotoxins. In a preferred embodiment, the chemokine is MIP-1xcex1, MIP-1xcex2, RANTES, MDC, I-309, eotaxin, MCP-3, or SDF-1.
Preferably the receptor ligand binding molecule is a natural or synthetic molecule that specifically binds the ligand, but is not the receptor or a fragment thereof. Examples of preferred receptor ligand binding molecules include polyanionic compounds, such as glycosaminoglycan. Preferred glycosaminoglycans are heparan, heparan sulfate, chondroitin sulfate, or dermatan sulfate. In yet another embodiment of the basic method of the invention, the receptor ligand and the receptor ligand binding molecule are noncovalently associated prior to administration to the patient to be treated. In other embodiments of the invention, the receptor ligand and the receptor ligand molecule are covalently associated prior to administration to the patient. In preferred embodiments, the composition resulting from combination of the receptor ligand and the receptor ligand binding molecule has a longer half-life than that of the receptor ligand alone.
Given their novel properties, receptor ligands in association with receptor ligand binding molecules should prove to be effective therapeutics. In addition, since the complexes are unable to trigger receptors, they should prove to be free from undesirable side effects resulting from the continued activation of their target receptor as has been observed in the use of chemokines to block HIV infection. Moreover, such complexes are unlikely to be deposited on irrelevant extracellular surfaces and therefore should specifically localize at appropriate in vivo locations to mediate a prophylactic or therapeutic effect.
In another aspect, the invention features novel methods for treating diseases or conditions, which are caused by or contributed to by the function of a ligand/receptor-mediated signaling pathway or which are dependent upon the extracellular recognition of a receptor by an infectious agent based on administration to a subject of a therapeutically effective amount of a receptor ligand and a receptor ligand binding molecule, wherein the receptor ligand and receptor ligand binding molecule are complexed or are capable of complexing in vivo, thereby antagonizing the function of the receptor or altering the extracellular recognition of the receptor by the infectious agent, resulting in treatment of the disease or condition.
In certain embodiments of the present invention, the disease or condition is dependent upon the extracellular recognition of a receptor by an infectious agent. In preferred embodiments, the infectious agent is a virus selected from the group comprising: an Human Immunodeficiency Virus such as HIV-1, an Epstein-Barr Virus, a Rhinovirus, a Poliovirus, a Rabies Virus, a Reovirus, an Influenza Virus, an Herpes Simplex Virus, an Hepatitis virus, a Togavirus, a Varicella-Zoster Virus, a Paramyxovirus, a Cytomegalovirus, a Subacute Sclerosing Panencephalitis Virus, an Adenovirus, a Poxvirus, a Reovirus, a Papovavirus, a Papillomavirus, a Polyomavirus, and a Slow virus.
In another embodiment, the infectious agent is a microbe which requires a specific host receptor or receptors for colonization or penetration. In preferred embodiments, the microbe is a bacterium selected from the group comprising: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, Mycobacterium gordonae, Mycobacterium leprae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis. Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus bovis, Streptococcus anginosus, Streptococcus pneumoniae, pathogenic Campylobacter species, pathogenic Enterococcus species, Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, pathogenic Bacteroides fragilis group species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema palladium, Treponema pertenue, Leptospira, and Actinomyces isrealli. In yet other embodiments, the microbe is a fungus selected from the group comprising: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatidis, Chlamydia trachomatis, and Candida albicans. 
In further embodiments of the invention, the disease or condition is caused by or contributed to by the function of a ligand/receptor-mediated signaling pathway. In one preferred embodiment, the disease or condition is, an inflammatory or an immune disease or disorder. In another preferred embodiment, the disease or condition is a cancer.
In still a further aspect, the invention features screening assays for identifying therapeutically effective formulations of particular receptor ligand-containing antagonist complexes for use e.g. in treating and/or preventing the development of a disease or condition that is caused by or contributed to by the function of a cell surface receptor.
Other features and advantages of the invention will be apparent from the following detailed description and claims.